The advent of recombinant DNA technology and genetic engineering has led to numerous efforts to develop methods that facilitate the transfection of therapeutic and other nucleic acid-based agents to specific cells and tissues. Known techniques provide for the delivery of such agents, including a variety of genes that are carried in recombinant expression constructs. These constructs are capable of mediating expression of the genes once they arrive within a cell. Such developments have been critical to many forms of molecular medicine, specifically gene therapy, whereby a missing or defective gene can be replaced by an exogenous copy of the functional gene.
Typically, nucleic acids are large, highly polar molecules. As such, nucleic acids face the impermeable barrier of the cellular membrane in eukaryotes and prokaryotes. The cell membrane acts to limit or prevent the entry of the nucleic acid into the cell. The development of various gene delivery methods has paralleled currently known gene therapy protocols. While much progress has been made in increasing the efficiency of gene delivery into cells, limited nucleic acid uptake or transfection remains a hindrance to the development of efficient gene therapy techniques.
Common approaches for delivering a nucleic acid into a cell include ex vivo and in vivo strategies. In ex vivo gene therapy methods, the cells are removed from the host organism, such as a human, prior to experimental manipulation. These cells are then transfected with a nucleic acid in vitro using methods well known in the art. These genetically manipulated cells are then reintroduced into the host organism. Alternatively, in vivo gene therapy approaches do not require removal of the target cells from the host organism. Rather, the nucleic acid may be complexed with reagents, such as liposomes or retroviruses, and subsequently administered to target cells within the organism using known methods. See, e.g., Morgan et al., Science 237:1476, 1987; Gerrard et al., Nat. Genet. 3:180, 1993.
Several different methods for transfecting cells can be used for either ex vivo or in vivo gene therapy approaches. Known transfection methods may be classified according to the agent used to deliver a select nucleic acid into the target cell. These transfection agents include virus dependent, lipid dependent, peptide dependent, and direct transfection (xe2x80x9cnaked DNAxe2x80x9d) approaches. Other approaches used for transfection include calcium co-precipitation and electroporation.
Viral approaches use a genetically engineered virus to infect a host cell, thereby xe2x80x9ctransfectingxe2x80x9d the cell with an exogenous nucleic acid. Among known viral vectors are recombinant viruses, of which examples have been disclosed, including poxviruses, herpesviruses, adenoviruses, and retroviruses. Such recombinants can carry heterologous genes under the control of promoters or enhancer elements, and are able to cause their expression in vector-infected host cells. Recombinant viruses of the vaccinia and other types are reviewed by Mackett et al., J. Virol. 49:3, 1994; also see Kotani et al., Hum. Gene Ther. 5:19, 1994.
However, viral transfection approaches carry a risk of mutagenicity due to possible viral integration into the cellular genome, or as a result of undesirable viral propagation. Many studies in vertebrate systems have established that insertion of retroviral DNA can result in inactivation or ectopic activation of cellular genes, thereby causing diseases. For a review, see Lee et al., J. Virol. 64:5958-5965, 1990. For example, one well known consequence of retroviral integration is activation of oncogenes. One study describes the activation of a human oncogene by insertion of HIV. Shiramizu et al., Cancer Res., 54:2069-2072, 1994. Viral vectors also are susceptible to interference from the host immune system.
Non-viral vectors, such as liposomes, may also be used as vehicles for nucleic acid delivery in gene therapy. In comparison to viral vectors, liposomes are safer, have higher capacity, are less toxic, can deliver a variety of nucleic acid-based molecules, and are relatively nonimmunogenic. See Felgner, P. L. and Ringold, G. M., Nature 337, 387-388, 1989. Among these vectors, cationic liposomes are the most studied due to their effectiveness in mediating mammalian cell transfection in vitro. One technique, known as lipofection, uses a lipoplex made of a nucleic acid and a cationic lipid that facilitates transfection into cells. The lipid/nucleic acid complex fuses or otherwise disrupts the plasma or endosomal membranes and transfers the nucleic acid into cells. Lipofection is typically more efficient in introducing DNA into cells than calcium phosphate transfection methods. Chang et al., Focus 10:66, 1988. However, some of the lipid complexes commonly used with lipofection techniques are cytotoxic or have undesirable non-specific interactions with charged serum components, blood cells, and the extracellular matrix. Furthermore, these liposome complexes can promote excessive non-specific tissue uptake.
One known protein dependent approach involves the use of polylysine mixed with a nucleic acid. The polysine/nucleic acid complex is then exposed to target cells for entry. See, e.g., Verma and Somia, Nature 389:239, 1997; Wolff et al., Science 247:1465, 1990. However, protein dependent approaches are disadvantageous because they are generally not effective and typically require chaotropic concentrations of polylysine.
xe2x80x9cNakedxe2x80x9d DNA transfection approaches involve methods where nucleic acids are administered directly in vivo. See U.S. Pat. No. 5,837,693 to German et al. Administration of the nucleic acid could be by injection into the interstitial space of tissues in organs, such as muscle or skin, introduction directly into the bloodstream, into desirable body cavities, or, alternatively, by inhalation. In these xe2x80x9cNakedxe2x80x9d DNA approaches, the nucleic acid is injected or otherwise contacted with the animal without any adjuvants, such as lipids or proteins, which typically results in only moderate levels of transfection, and the insufficient expression of the desired protein product. It has recently been reported that injection of free (xe2x80x9cnakedxe2x80x9d) plasmid DNA directly into body tissues, such as skeletal muscle or skin, can lead to protein expression, but also to the induction of cytotoxic T lymphocytes and antibodies against the encoded protein antigens contained in the plasmid. See Ulmer et al., Science, 259, 1993, 1745-1749; Wang et al., Proc. Nat. Acad. Sci. U.S.A. 90, 4157-4160, 1993; Raz et al., Proc. Nat. Acad. Sci. U.S.A. 91, 9519-9523, 1994.
Electroporation is another transfection method. See U.S. Pat. No. 4,394,448 to Szoka, Jr., et al. and U.S. Pat. No. 4,619,794 to Hauser. The application of brief, high-voltage electric pulses to a variety of animal and plant cells leads to the formation of nanometer-sized pores in the plasma membrane. DNA can enter directly into the cell cytoplasm either through these small pores or as a consequence of the redistribution of membrane components that accompanies closure of the pores. The use of electroporation as a tool to deliver DNA into cells has had limited success for in vivo applications.
A common disadvantage to known non-viral nucleic acid delivery techniques is that the amount of exogenous protein expression produced relative to the amount of exogenous nucleic acid administered remains too low for most diagnostic or therapeutic procedures. Low levels of protein expression are often a result of a low rate of transfection of the nucleic acid or the instability of the nucleic acid.
Despite numerous research efforts directed at finding efficient methods for nucleic acid delivery, most known techniques fail to result in sufficient cell transfection to achieve the desired protein expression. There is still a need to develop a nucleic acid delivery method that efficiently introduces recombinant expression constructs encoding useful genes into cells, while minimizing undesirable effects.
The present invention describes methods for introducing nucleic acids into a target cell using a transition metal enhancer. In accordance with the methods of the present invention, a mixture containing the nucleic acid and a transition metal enhancer is exposed to cells. The nucleic acid is then taken up into the interior of the cell with the aid of the transition metal enhancer. Since nucleic acids can encode a gene, the method can be used to replace a missing or defective gene in the cell. The method can also be used to deliver exogenous nucleic acids operatively coding for polypeptides that are secreted or released from target cells, thus resulting in a desired biological effect outside the cell. Alternatively, the methods can be used to deliver exogenous nucleic acids into a target cell that are capable of regulating the expression of a predetermined endogenous gene. This can be accomplished by encoding the predetermined endogenous gene on the nucleic acid or by encoding the nucleic acid with a sequence that is the Watson-Crick complement of the mRNA corresponding to the endogenous gene.
In particular, the present invention relates to a method for delivering a nucleic acid into a target cell by contacting a cell with a solution containing a nucleic acid and a transition metal enhancer. The cell may be derived from or contained within an organism or a primary cell culture. The nucleic acid sequence to be delivered is normally determined prior to use of the disclosed method. In some embodiments, a nucleic acid is delivered to a target cell by contacting a cell with a solution containing a nucleic acid, a transition metal enhancer and cationic lipid.
In one embodiment, the solution that facilitates intracellular delivery of therapeutically effective amounts of nucleic acid to target cells may be suitable for use with a variety of cell types including, but not limited to, those associated with the various secretory glands (e.g., mammary, thyroid, pancreas, stomach, and salivary glands), musculature connective tissue, bone, bladder, skin, liver, lung, kidney, the various reproductive organs such as testes, uterus and ovaries, nervous system, all other epithelial, endothelial, and mesodermal tissues.
In other embodiments, the transition metal enhancer is a complex, adduct, cluster or salt of a d-block element, a lanthanide, aluminum, and/or gallium. In yet other embodiments, the transition metal enhancer is a zinc, nickel, cobalt, copper, aluminum, or gallium complex.
The present invention provides a novel method for delivering a nucleic acid into a target cell. In accordance with the methods of the present invention, the nucleic acid and transition metal enhancer are exposed to cells. When the nucleic acid encodes a useful protein, the exposure may result in measurable expression of the protein. Such protein expression is useful in the practice of both diagnostic and therapeutic strategies.